To develop a murine model system to test the role of monocyte-derived macrophages in atherosclerosis, the osteopetrotic (op) mutation in the macrophage colonystimulating factor gene was bred onto the apolipoprotein E (apoE)-deficient background. The doubly mutant (op/apoEdeficient) mice fed a low-fat chow diet had significantly smaller proximal aortic lesions at an earlier stage of progression than their apoE-deficient control littermates. These lesions in the doubly mutant mice were composed of macrophage foam cells. The op/apoE-deficient mice also had decreased body weights, decreased blood monocyte differentials, and increased mean cholesterol levels of 41300 mg/dl. Statistical analysis determined that atherosclerosis lesion area was significantly affected by the op genotype and gender. The confounding variables of body weight, plasma cholesterol, and monocyte differential, which were all affected by op genotype, had no significant additional effect on lesion area once they were adjusted for the effects of op genotype and gender.

RAW 264 mouse macrophage cells were stably transfected with human apolipoprotein E (apoE) expression vectors. Clonal derivatives were characterized for expression of the human apoE2, apoE3, and apoE4 isoforms. An apoE4-expressing clonal cell line and a non-expressing clonal control cell line were loaded overnight with either The 8-Br-cAMP-mediated increase in cholesterol efflux was also observed in non-apoE-secreting cells incubated with exogenous apoE or apoAI, and the effect of apoE was saturable. The apoE2, apoE3, and apoE4 isoforms were equally efficient in promoting 8-BrcAMP-dependent cholesterol efflux. Reductive methylation of apoE abolished its ability to promote 8-Br-cAMPdependent cholesterol efflux. Brefeldin A and monensin, inhibitors of protein processing through the Golgi, both blocked the 8-Br-cAMP stimulation of cholesterol efflux to exogenous apoE. 8-Br-cAMP induced specific apoE and apoAI binding, but not apoE degradation, by the RAW cells. We present a model wherein cAMP induces a membrane apolipoprotein receptor that does not lead to endocytosis and degradation, but instead promotes the transfer of lipids to apolipoproteins, which can then be released from the cell.

No abstract

No abstract

Transgenic and gene knockout techniques allow for in vivo study of the consequences of adding or subtracting specific genes. However, in some instances, such as the study of lethal mutations or of the physiological consequences of changing gene expression, turning on and off an introduced gene at will would be advantageous. We have used cytochrome p450 1A1 promoter to drive expression of the human apolipoprotein E (apoE) gene in transgenic mice. In six independent lines, robust expression of the transgene depended upon injection of the inducer beta-naphthoflavone, whereas the seventh line had high basal expression that was augmented further by the inducer. The low level of basal expression in an inducer-dependent line was confirmed upon breeding the transgene onto the hypercholesterolemic apoE-deficient background. In the basal state transgene expression was physiologically insignificant, as these mice were as hypercholesterolemic as their nontransgenic apoE-deficient littermates. When injected with the inducer, plasma cholesterol levels of the transgenic mice decreased dramatically as apoE expression was induced to yield greater than physiological levels in plasma. The inducer could pass transplacentally from an injected mother to her fetuses with concomitant induction of fetal transgene mRNA. Inducer could also pass via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma. These finding suggest a strategy to temporarily ameliorate genetic deficiencies that would otherwise lead to fetal or neonatal lethality.